Materials Today Communications 14 (2018) 106–115

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Materials Today Communications

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Nanopesticidal effects of Pongamia pinnata leaf extract coated zinc oxide T

nanoparticle against the Pulse beetle, Callosobruchus maculatus
⁎
Balasubramanian Malaikozhundana, , Jayaraj Vinodhinib
a
Biomaterials and Biotechnology in Animal Health Lab, Department of Animal Health and Management, Alagappa University, Science Campus 6th Floor, Burma Colony,
Karaikudi, 630 004, Tamil Nadu, India
b
Department of Biotechnology, Dr.Umayal Ramanathan College for Women, Karaikudi, 630 003, Tamil Nadu, India

A R T I C L E I N F O A B S T R A C T

Keywords: Callosobruchus sp. is the major cause of damage to stored pulses. Recent advances in nanotechnology have
Pongam provided us a promising tool for the management of insect pest of essential commodities. In the present study,
Zinc oxide we report the green synthesis and biological evaluation of Pongamia pinnata leaf extract coated zinc oxide na-
Nanoparticle noparticles (Pp-ZnO NPs) on the pulse beetle, C. maculatus. The green synthesized Pp-ZnO NPs were bio-phy-
Green pesticides
sically characterized by UV–vis spectroscopy, X-ray diffraction (XRD), Fourier transform infrared spectroscopy
Callosobruchus maculatus
(FTIR), Transmission electron microscopy (TEM), Selective area electron diffraction (SAED), Energy dispersive
X-ray (EDX) analysis and zeta potential. The bio-physical characterization revealed that the Pp-ZnO NPs has a
hexagonal wurtzite structures with a mean particle size of 21.3 nm. In addition, zeta potential measurement
demonstrated that the Pp-ZnO NPs are negatively charged (−12.45 mV) and are moderately stable. The pesti-
cidal effect of Pp-ZnO NPs was tested against the pulse beetle, C. maculatus. Pp-ZnO NPs reduced the fecundity
(eggs laid) and hatchability of C. maculatus in a dose-dependent manner. A significant delay in the larval, pupal
and total development period of C. maculatus was observed after treatment with Pp-ZnO NPs at 25 μg mL−1.
Furthermore, Pp-ZnO NPs are more effective in the control of C. maculatus and caused 100% mortality at
25 μg mL−1. The LC50 value was estimated to be 10.85 μg mL−1. In addition, treatment with Pp-ZnO NPs de-
creased the mid-gut α-amylase, cysteine protease, α-glucosidase, β-glucosidase, glutathione S-transferase (GST)
and lipase activity in C. maculatus. This study concludes that Pp-ZnO NPs are effective against C. maculatus and
could be used as an alternative pest control agent in the management of stored grain insect pests in the future.

1. Introduction
Nanotechnology has provided new insight to prepare highly spe-
cialized nanoparticulates of any size and shape and have led to the
development of new biocidal agents. Metal oxide nanoparticles play an
important role in the elimination of hazardous chemicals from the en-
vironment [1]. Among the metal oxide nanoparticles, zinc oxide has
vast applications such as optical, piezoelectric, magnetic, and gas sen-
sing. Besides these properties, ZnO nanostructure exhibits high catalytic
and strong adsorption ability. ZnO NPs are more frequently used in the
manufacture of sunscreens [2], ceramics and rubber processing, was-
tewater treatment, and as a fungicide [3]. Several physical and che-
mical methods are used for the synthesis of metal nanoparticles but the
presence of toxic chemicals limits their application [4]. Currently,
biological synthesis of nanoparticle using plants or plant products is
gaining importance due to its simplicity, eco-friendliness and wide-
spread antimicrobial activity [5,6]. Biosynthesis of zinc oxide
nanoparticles using plants such as Aloe vera [7], Pongamia pinnata [8],
Laurus nobilis [9] and Plectranthus barbatus [10] has been reported.
In agriculture, nanomaterials help to develop effective methods for
insect pest management [11]. The toxic effects of silver, zinc, alu-
minum, and titanium oxide nanoparticles on insect pests have been
reported [12,13]. Among them, zinc oxide (ZnO) nanoparticle is re-
cognized as agricultural fungicide [14,15]. The biopesticidal effects of
Bacillus thuringiensis coated zinc oxide nanoparticles on the pulse beetle,
Callosobruchus maculatus F. (Coleoptera: Bruchidae) has been reported
earlier [16]. C. maculatus is the major storage pest of cowpea
throughout the world. The control of stored-grain insect pests largely
depends on chemical insecticides and fumigants. The large scale ap-
plication of chemical insecticides leads to the contamination of grains
with pesticide residues [17]. Moreover, the difficulty in controlling the
insect pests in stored grain is the resistance to chemical pesticides.
Therefore, there is an increasing demand to search for alternative
control measures that are highly effective and safe to humans.

⁎
Corresponding author.
E-mail address: kozhundan.malai@gmail.com (B. Malaikozhundan).

https://doi.org/10.1016/j.mtcomm.2017.12.015
Received 20 October 2017; Received in revised form 22 November 2017; Accepted 27 December 2017
Available online 30 December 2017
2352-4928/ © 2017 Elsevier Ltd. All rights reserved.
B. Malaikozhundan, J. Vinodhini
Botanical pesticides based on plant extracts or plant products have
been traditionally used as an alternative to conventional synthetic
pesticides. Furthermore, they are safer to the environment and non-
target organisms [18]. P. pinnata L., is a forest tree belonging to the
family Leguminosae and is commonly used for biodiesel production
[19]. Different parts such as leaves, root, bark, flowers and seeds of P.
pinnata contain a number of furano-flavonoid compounds which are
known to possess pesticidal activity. Karanjin, pongamol, pongapin,
glabrin, karanja chromene, karanjone and pongaglabrone are the
principal furano flavonoids present in the seed oil [20]. In the present
study, we report the biological synthesis and bio-physical character-
ization of zinc oxide nanoparticles using the leaf extracts of P. pinnata
(Pp-ZnO NPs). In addition, the pesticidal effects of Pp-ZnO NPs were
tested against the cowpea bruchid by investigating the mortality and
changes in the mid-gut digestive enzyme activities.
2. Materials and methods
2.1. Collection of plant material
P. pinnata leaves were collected from in and around the regions of
Karaikudi (North latitude between 9.43′ and 10.42, East longitude be-
tween 77.47′ and 78.49′), Tamil Nadu, India. The identification of the
plant was authenticated by the Botanical Survey of India. A voucher
specimen of the plant was preserved in the Department (voucher spe-
cimen DAHM-17-005). Fresh leaves were washed with distilled water,
followed by air drying and has been used for the present investigation.
2.2. Preparation of leaf extract of P. pinnata
Aqueous leaf extract of P. pinnata was prepared following the
method by Vijayakumar et al. [9]. Briefly, 50 g of washed and dried fine
leaves was placed in 250 mL glass beaker along with 100 mL of distilled
water. The mixture was boiled for 60 min in hot plate until the colour of
solution changes to light yellow. The extract was cooled, filtered and
then stored at 4 °C for further experiments.
2.3. Synthesis of zinc oxide nanoparticles using the leaf extract of P.
pinnata
The synthesis of zinc oxide nanoparticles using the aqueous leaf
extracts of P. pinnata (Pp-ZnO NPs) was carried out by the method of
Azizi et al. [21]. Zinc acetate dihydrate (99% purity) and sodium hy-
droxide pellet was used as the precursor material. Briefly, 20 mL of
0.02 M zinc acetate dihydrate was added to 50 mL of distilled water
with vigorous stirring. After 10 min of stirring, 250 mL of aqueous leaf
extract of P. pinnata and aqueous 2.0 M NaOH was added. This has
resulted in a white aqueous solution at pH 12. The contents were then
placed in magnetic stirrer for 2 h to obtain precipitate. The precipitate
was then taken out and washed repeatedly with distilled water, fol-
lowed by ethanol to remove the impurities of final product. Finally, a
white powder of ZnO nanoparticles was obtained after drying at 60 °C
in a vacuum oven over night.
2.4. Bio-physical characterization of zinc oxide nanoparticles
2.4.1. UV–vis spectroscopy
The reduction of Zn+ ions was monitored by measuring the ab-
sorption spectrum of the reaction medium after 30 min. About 1 mL of
the sample was collected for UV–vis spectrum analysis and the max-
imum absorbance spectrum of Pp-ZnO NPs was observed at
200–700 nm (Schimadzu UV-1800) [22].
2.4.2. X-ray diffraction (XRD) analysis
The particle size of Pp-ZnO NPs was determined using XRD 6000/
6100 (Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan). Briefly, the
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Materials Today Communications 14 (2018) 106–115
nanomaterial was finely ground, and the average bulk composition was
determined. The grain size of zinc oxide nanoparticles was determined
using Debye Scherrer’s equation.
D = 0.94λ/β cos θ
Where λ is the wavelength (Cu Kα), β is the full width half- maximum
(FWHM) of the ZnO (101) line and θ is the diffraction angle [23].
2.4.3. Fourier transform infrared (FTIR) spectroscopy
Two milligram of Pp-ZnO NPs was mixed with 200 mg potassium
bromide (FTIR grade) and pressed into a pellet. The pellet was placed
into the sample holder and FTIR spectra were recorded under FTIR
spectroscopy (Thermo Scientific Nicolet-iS5, Waltham, USA) at a re-
solution of 4 cm−1 [23].
2.4.4. High resolution transmission electron microscopy (HR-TEM) and
Selective area electron diffraction (SAED)
The morphology of Pp-ZnO NPs was examined under high resolu-
tion transmission electron microscopy (JOEL model instrument 1200
EX) with an acceleration voltage of 200 kV. A drop of sample (Pp-ZnO
suspension in methanol) was placed on a standard carbon coated
copper grid and was allowed to dry before recording the micrographs
[9]. TEM images were analyzed using JMicroVision code to determine
the particle size distribution of Pp-ZnO NPs. The J MicroVision code can
calculate the average diameter (d) of the particles in an image from any
one of their geometrical characteristics, namely area (d = 2p[s/p]),
perimeter (d = p/p), or average of longest and shortest diameters
(d = 0.5[d1 + d2]) in 2-D [24]. Selected area electron diffraction pat-
tern (SAED) was used to analyze the phase structure and crystallinity of
nanoparticles.
2.4.5. Energy dispersive X-ray (EDX) analysis
An energy-dispersive X-ray detection instrument (EDX) (Oxford
INCA 400) was used to examine the elemental composition of the
sample.
2.4.6. Zeta potential distribution
Zeta potential was determined using a Zeta sizer Nano Z system
(Malvern Instruments, UK) by dispersing the synthesized Pp-ZnO NPs in
water [16].
2.5. Pesticidal activity of Pp-ZnO NPs
2.5.1. Culture maintenance of C. maculatus
Adult C. maculatus and green gram (Vigna radiata) seeds were ob-
tained from the National Pulses Research Centre, Vamban, Tamil Nadu,
India. The culture was reared on healthy, sterilized seeds of green gram,
V. radiata and maintained at 30 ± 1 °C, 60 ± 5% relative humidity
(RH) and 2 h light: 12 h dark period for three generations before ex-
perimentation to ensure that they were genetically and phenotypically
alike. The beetles were grown under moderately crowded conditions to
ensure proper development with equal size of the resultant adults and
used as a source for bioassay [25].
2.5.2. Insect bioassay
The efficacy of Pp-ZnO NPs against C. maculatus was tested at five
different concentrations (5, 10, 15, 20 and 25 μg mL−1). About 25 seeds
of V. radiata were dipped in different concentrations of Pp-ZnO NPs, air
dried and kept in a plastic jar. Sterilized untreated seeds were used as
control. Ten newly emerged males and females (1:1) of C. maculatus
were introduced in pairs into the plastic jar and covered with muslin
cloth. Introduced pairs were allowed to oviposit on the treated seeds of
V. radiata. Five replicates were performed for each concentration of Pp-
ZnO NPs. Bioassay was maintained at a controlled conditions of
30 ± 1 °C and 60 ± 5% RH. The fecundity (number of eggs laid),
B. Malaikozhundan, J. Vinodhini
percentage hatchability of eggs, grub (larval) period, pupal period, total
development period and mortality of C. maculatus were determined for
each concentration of Pp-ZnO NPs as described below. The pesticidal
effects of Pp-ZnO NPs were compared with leaf extract and uncoated
ZnO NPs at 25 μg mL−1.
2.5.3. Development of C. maculatus
Fecundity was determined by counting the number of eggs laid by
individual female at an interval of 24 h from the day of emergence until
death. If oviposition had not occurred, another virgin pair was replaced.
The total number of adults emerged in each replication was calcu-
lated by counting the number of adults emerged twice a day from
morning and evening till the last emergence.
Hatchability of eggs in each replication was determined using the
following formula
Number of eggs hatched
Hatchability(%) = × 100
Number of eggs laid
The duration of larval and pupal stages was recorded from the time
of egg hatching until the emergence of adults. The total developmental
period of C. maculatus was determined by counting the days between
the oviposition and the adult emergence in each replication.
2.5.4. Mortality of C. maculatus
Mortality of C. maculatus was determined by counting the number of
dead beetles in each treatment at regular intervals (24 h). The percen-
tage of mortality was calculated using Abbott’s formula [26], while
LC50was calculated using probit analysis according to Finney [27].
2.6. Sample preparation and mid-gut digestive enzyme activity
Ten larvae that emerged from the treated seeds of V. radiata were
pooled from each replicate and homogenized with PBS buffer (100 mM
sodium phosphate and 500 mM sodium chloride, pH 7.6) in a ratio of
100 μL/larva. The samples were agitated for 30 min at 4 °C and then
centrifuged at 1700g for 5 min. The resulting supernatant was used for
α-amylase, cysteine protease and glutathione-S- transferase (GST) as-
says.
For α- and β-glucosidase activity assay, larvae were homogenized in
distilled water, subjected to freeze and thaw cycles in liquid nitrogen
thrice. The samples were centrifuged at 18,000g for 30 min at 4 °C.
After centrifugation, the supernatant was discarded and the pellet was
resuspended in a buffer containing 0.1% Nonidet P-40 (NP-40), 20 mM
sodium phosphate (pH 7.4), 0.5 mM imidazole, 1 mM PMSF (phenyl
methane sulfonyl fluoride) and 1 mM benzamidine. The suspension was
agitated overnight at 4 °C, followed by centrifugation at 18,000g for
30 min. The final supernatant was used for α-glucosidase assay.
2.6.1. α-Amylase activity
α-amylase activity assay was carried out according to de Sa et al.
[28]. Briefly, 1% (w/v) soluble starch was used as substrate. Forty-four
microliter of sample was added to 6 μL of 1% starch or water and in-
cubated in a water bath at 37 °C for 45 min. The reaction was stopped
by adding 3, 5-dinitrosalicylic acid (DNS) followed by incubation in a
boiling water bath for 5 min. Absorbance was measured at 540 nm. A
standard curve of α-amylase absorbance against the amount of maltose
released was used to quantify the enzyme activity. One unit of α-
amylase activity was defined as the enzyme quantity that increased the
absorbance by 0.1 units over 30 min.
2.6.2. Cysteine protease activity
Cysteine protease activity was determined according to de Sa et al.
[28] by using azocasein as substrate. Azocasein was prepared as 1% (w/
v) solution in citrate–phosphate buffer containing 100 mM sodium ci-
trate (pH 5.6), 100 mM sodium phosphate, 0.1% Triton X-100 and
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Materials Today Communications 14 (2018) 106–115
1.5 mM 1, 4-dithioerythritol (DTT). Thirty microliter of larval extract
was incubated with 80 μL of 1% azocasein and 10 μL of citrate–pho-
sphate buffer, at 37 °C for 1 h. Proteolysis was stopped by adding 300 μL
of 10% trichloroaceticacid (TCA). The reaction mixture was then cen-
trifuged at 20,000g for 15 min at 4 °C. The supernatant was collected
and 300 μL of 1 M NaOH was added to each sample. The absorbance
was read at 440 nm and cysteine protease activity was calculated ac-
cording to a standard curve derived from pure papain (Sigma)
(0.5–11 μg/mL) and azocasein as substrate.
2.6.3. α- and β-glucosidase activity
α and β-glucosidase activities were measured according to the
procedure of Khosravi and Sendi [29] using pNαG (p-Nitrophenyl-α-D-
glucopyranoside) and pNβG (p-Nitrophenyl-β-D-glucopyranoside) as
substrates. Briefly, 10 μL of the homogenate was incubated with 45 μL
of substrate (25 mM) and 115 μL of glycine phosphate-acetic-citric
buffer (40 mM) at 37 °C for 30 min. The reaction was stopped by the
addition of 600 μL of NaOH (0.25 M). The change in the absorbance was
read at 405 nm using a microplate reader (Robonik, USA) after 10 min.
A standard curve of absorbance against the amount of p-nitrophenol
released was constructed to calculate the amount α & β-glucosidases.
2.6.4. Glutathione transferase (GST) activity
GST activity was performed with seven substrates [1-chloro-2,4-
dintrobenzene (CDNB), 4-chloro-7-nitrobenzo-2-oxa-1,3 diazole, p-ni-
trophenyl-acetate, ethacrynic acid, 1,2-dichloro-4-nitrobenzene, 4-hyr-
oxylnonenal and p-nitrophenychloride] according to the procedures of
Nay et al. [30]. One unit of enzyme activity was defined as the amount
of enzyme that catalyzed the formation of 1 μmole of product/min at
25 °C in the optimal assay condition for each substrate. The specific
activity was expressed in μmole of product/min/mg protein. All initial
rates were corrected for the background non-enzymatic reaction and
were completed in triplicate. A Shimadzu UV-1800 double beam
spectrophotometer was used for enzymatic and protein assay.
2.6.5. Lipase activity
Lipase activity was determined following the procedure of Khosravi
and Sendi [29]. Briefly, 10 μL of mid-gut tissue extracts was mixed with
18 μL of 50 mM p-nitrophenyl butyrate (substrate) and 172 μL of 1 M
universal buffer solution (pH 7). It was then incubated at 37 °C and the
absorbance was read at 405 nm. One unit of enzyme releases 1.0 nmol
of p-nitrophenyl per minute at pH 7.2, at 37 °C using pnitrophenyl
butyrate as the substrate.
2.6.6. Determination of protein concentration
Protein concentration in each larval extract was determined using
BIORAD protein assay kit (Bio-Rad, USA,) according to the method of
Bradford [31]. Bovine serum albumin (BSA) was used as a standard.
2.7. Statistical analysis
Data was analyzed using one-way analysis of variance (ANOVA)
followed by Tukey’s HSD test. The results were expressed as mean ±
standard deviations (SD) and considered statistically significant at
P < 0.05.
3. Results
3.1. Bio-physical characterization of Pp-ZnO NPs
The synthesis of Pp-ZnO NPs was confirmed by recording the UV–vis
absorbance spectrum from the wavelength range of 200–700 nm. An
absorption peak (λmax) at 370 nm characteristic of ZnO NPs was ob-
served for Pp-ZnO NPs (Fig. 1A).
The XRD spectrum reveals that the synthesized Pp-ZnO NPs were
pure and crystalline in nature. The Bragg’s reflection peaks at 31.62°,
B. Malaikozhundan, J. Vinodhini Materials Today Communications 14 (2018) 106–115

Fig. 1. Bio-physical characterization of Pp-ZnO NPs. (A) UV–vis absorbance spectrum at different wavelengths (B) XRD spectrum showing various Bragg’s reflection peaks (C) FTIR
spectra showing functional molecules (D) TEM image (a) and SAED pattern (b) (E) EDX showing the elemental composition and (F) Zeta potential measurement (a) P. pinnata leaf extract
(b) Pp-ZnO NPs.

34.88°, 36.50°, 39.34°, 47.24°, 57.24°, 63.85°, 66.26° and 69.27° cor-
responds to the lattice planes at (100), (002), (101), (102), (110),
(103), (200) and (112) reflection lines of hexagonal ZnO NPs, respec-
tively. XRD pattern reflects the hexagonal wurtzite structure of Pp-ZnO
NPs (Fig. 1B). The average crystalline size of Pp-ZnO NPs was found to
be 19.4 nm using Debye Scherrer equation.
The possible biomolecules present in the P. pinnata leaf extract and
its interaction with 0.1 M zinc acetate were characterized by FTIR
spectroscopy. The FTIR spectra of synthesized Pp-ZnO NPs exhibited
relatively a high intensity FTIR band compared to leaf extract (Fig. 1C).
The FTIR spectra of Pp-ZnO NPs exhibited peaks at 1754, 1655, 1639,
1390, 1148 and 1086 cm−1. The intense broad band at 1754 and
1655 cm−1could be attributed to the stretching of polyphenols. The
band observed at 1639 and 1390 cm−1 could be due to the stretching of
aromatic amines. The band at 1148 cm−1 corresponds to CeC
stretching vibrations of alkanes. Band at 1086 cm−1 could be attributed

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to CeN stretching vibrations of aliphatic amides. The FTIR spectra of P.

pinnata leaf extract showed intense broad band at 1735 cm−1 which
corresponds to hydroxyl groups. The band at 1640 and 1436 cm−1 was
attributed to the CeOH group of alkanes. The band observed at 1250
and 1132 cm−1 could be due to the vibrations of HeH and OH groups
respectively. The band at 1065 cm−1 could be due to the stretching of
carboxyl groups.
The size and structural morphology of biologically synthesized Pp-
ZnO NPs are further confirmed by HR-TEM analysis. The results re-
vealed that Pp-ZnO NPs are agglomerated and exhibited a hexagonal
wurtzite structure (Fig. 1Da). The particle size of Pp-ZnO NPs ranged
from 26.5, 21.4 and 16.1 nm by area, perimeter and dimensions re-
spectively with a mean size of 21.3 nm. SAED pattern also revealed the
hexagonal wurtzite structure and crystalline nature of Pp-ZnO NPs
(Fig. 1Db).
EDX analysis revealed that the elemental composition of zinc was
87.36 (Fig. 1E). The zeta potential of leaf extract and Pp-ZnO NPs are
shown in Fig. 1FA and 1FB, respectively. The zeta potential value of
−12.45 mV reveals that Pp-ZnO NPs are negatively charged and are
moderately stable.

3.2. Effect of Pp-ZnO NPs on the development of C. maculatus

Treatment with Pp-ZnO NPs reduced the number of eggs laid (fe-
cundity) by C. maculatus in a dose-dependent manner (Fig. 2A). The
number of eggs laid was reduced to 88% at 5 μg mL−1 of Pp-ZnO NPs.
However, Pp-ZnO NPs at 25 μg mL−1 significantly reduced the number
of eggs laid to 24%. On the other hand, the number of eggs laid was
reduced to 65% and 42% at 25 μg/mL of uncoated ZnO NPs and leaf

Fig. 3. Effect of Pp-ZnO NPs on the larval development period (A), pupal development
period (B) and (C) total development period of C. maculatus. Each bar indicated
mean ± SD of three replications. Bars not labeled by the same letter represent statistical
significance at p < 0.05 using ANOVA followed by Tukey’s HSD test.

Fig. 2. Effect of Pp-ZnO NPs on the fecundity (A) and hatchability of C. maculatus (B).
Each bar indicated mean ± SD of three replications. Bars not labeled by the same letter
represent statistical significance at p < 0.05 using ANOVA followed by Tukey’s HSD test.
extract, respectively.
The hatchability of eggs was significantly reduced in all the treat-
ment groups compared to control. Treatment with 25 μg mL−1 of un-
coated ZnO NPs and leaf extract reduced the hatchability to 72% and
64%, respectively. However, hatchability was severely dropped to 30%
after treatment with 25 μg mL−1of Pp-ZnO NPs (Fig. 2B).
In control, the duration of development of C. maculatus larva was
21 days. The larval development period was increased to 22 days and
24 days after treatment with 25 μg mL−1of uncoated ZnO NPs and leaf
extract, respectively. However, a prolonged larval period (26 days) was
observed after treatment with 25 μg mL−1 of Pp-ZnO NPs (Fig. 3A).
Similar to these observations, the pupal period was increased to 9 days
and 10 days after treatment with 25 μg mL−1 of uncoated ZnO NPs and
leaf extract, respectively. Treatment with 25 μg mL−1 of Pp-ZnO NPs
prolonged the pupal period to 11 days compared to control (Fig. 3B).
A significant delay in the total development of C. maculatus was
observed following treatment with Pp-ZnO NPs compared to control.
The total development period of C. maculatus was 37 days after treat-
ment with Pp-ZnO NPs at 25 μg mL−1. On the other hand, the total

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Table 1
Probit analysis for the estimation of LC50 of Pp-ZnO NPs against C. maculatus.

Treatment χ2- values for Heterogeneity* Regression equations LC50 (μg mL-1) 95% Confidence limits

Lower Upper

ZnO NPs 4.114 Y = 4.22 + 0.41X 40.50 36.28 62.32

Leaf extract 2.145 Y = 4.06 + 0.38X 14.10 10.20 22.35
Pp-ZnO NPs 0.850 Y = 4.14 + 0.24X 10.85 7.46 14.05

*
χ2 = Chisquare value significant at P < 0.05; LC50 = Lethal concentration for 50% mortality.

development period was 31 days and 34 days following treatment with
25 μg mL−1 of uncoated ZnO NPs and leaf extract, respectively
(Fig. 3C).
3.3. LC50 o f Pp-ZnO NPs on C. maculatus
The LC50 values for uncoated ZnO NPs, leaf extract and Pp-ZnO NPs
on the mortality of C. maculatus calculated using probit analysis are
presented in Table 1. It was evident that C. maculatus was more sensi-
tive to Pp-ZnO NPs than leaf extract and uncoated ZnO NPs. The LC50
value of Pp-ZnO NPs was 10.85 μg mL−1. However, the LC50 values of
uncoated ZnO NPs and leaf extract were 40.5 and 14.10 μg mL−1, re-
spectively (Fig. 4A–C).
3.4. Effect of Pp-ZnO NPs on the mortality of C. maculatus
Mortality of C. maculatus treated with uncoated ZnO NPs, leaf ex-
tract and Pp-ZnO NPs are presented in Fig. 4D. Mortality of C. maculatus
increased with increasing concentration of Pp-ZnO NPs
(5–25 μg mL−1). The mortality was 30% at 5 μg mL−1 and it was sig-
nificantly increased to 100% at 25 μg mL−1 of Pp-ZnO NPs. Mortality
was 30% and 82% after treatment with 25 μg mL−1 of uncoated ZnO
NPs and leaf extract, respectively.
3.5. Effect of Pp-ZnO NPs on the mid-gut digestive enzyme activity of C.
maculatus
No differences in the α-amylase activity of C. maculatus were ob-
served in uncoated ZnO NPs and leaf extract treated groups (0.9 and
0.8 μg maltose/mg larva/min, respectively) at 25 μg mL−1. However,
significant reduction in the α-amylase activity of C. maculatus was ob-
served after treatment with Pp-ZnO NPs at 25 μg mL−1 (0.18 μg mal-
tose/mg larva/min) compared to control (Fig. 5A). Cysteine protease
activity was dramatically decreased in C. maculatus treated with Pp-
ZnO NPs at 25 μg mL−1 (1.0 μg/mg larva) compared to control. No
significant differences in the cysteine protease activity were observed in
C. maculatus treated with uncoated ZnO NPs and leaf extract (2.1 and
2.0 μg/mg larva, respectively) (Fig. 5B). The α-glucosidase activity of C.

Fig. 4. Lethal concentration showing 50% mortality of C. maculatus treated with uncoated ZnO NPs (A), leaf extract (B) and Pp-ZnO NPs (C). Mortality of C. maculatus treated with
different concentrations of Pp-ZnO NPs (D). Each bar indicated mean ± SD of three replications. Bars not labeled by the same letter represent statistical significance at p < 0.05 using
ANOVA followed by Tukey’s HSD test.

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Fig. 5. Physiological effect of Pp-ZnO NPs on the mid-gut digestive enzymes of C. maculatus (A) α-amylase activity (B) cysteine protease activity (C) α- glucosidase activity (D) β-
glucosidase activity (E) Glutathione S-transferase (GST) activity and (F) lipase activity. Each bar indicated mean ± SD of three replications. Bars not labeled by the same letter represent
statistical significance at p < 0.05 using ANOVA followed by Tukey’s HSD test.

maculatus in the control group was 7.8 mU/mg larva. Reduction in the
α-glucosidase activity was observed in C. maculatus treated with un-
coated ZnO NPs and leaf extract (7.6 and 7.2 mU/mg larva, respec-
tively) at 25 μg mL−1. On the other hand, the α-glucosidase activity was
significantly reduced (4.2 mU/mg larva) after treatment with Pp-ZnO
NPs at 25 μg mL−1 (Fig. 5C). Similarly, a greater reduction in the β-
glucosidase activity of C. maculatus was observed after treatment with
Pp-ZnO NPs at 25 μg mL−1 (0.2 mU/mg larva) (Fig. 5D).Treatment with
Pp-ZnO NPs at 25 μg mL−1 reduced the GST activity (4.7 μmol/mg/
min) in C. maculatus compared to control (8.0 μmol/mg/min). GST
activity of C. maculatus treated with uncoated ZnO NPs and leaf extract
was 7.8 and 7.1 μmol/mg/min, respectively (Fig. 5E). Lipase activity
was significantly inhibited in C. maculatus after treatment with Pp-ZnO
NPs at all concentrations tested. The lipase activity of C. maculatus
treated with uncoated ZnO NPs and leaf extract was 0.09 and
0.08 μmol/mg/min, respectively. However, it was greatly reduced to
0.01 μmol/mg/min at 25 μg mL−1 of Pp-ZnO NPs (Fig. 5F).
3.6. Estimation of protein in the mid-gut digestive enzymes of C. maculatus
The amount of protein in the mid-gut amylase, cysteine protease, α-
and β-glucosidases, glutathione transferase (GST) and lipase and their
specific activity are presented in Table 2.
4. Discussion
Nanotechnology has a prominent place in all fields of science. Zinc
oxide nanoparticles are one of the most versatile materials, due to their
diverse properties, functionalities, and applications. ZnO NPs have
tremendous physical and optical properties. Zinc oxide nanoparticles
can be synthesized by chemical methods but recently due to the evo-
lution of green chemistry, the synthesis of ZnO NPs by biological

112
B. Malaikozhundan, J. Vinodhini Materials Today Communications 14 (2018) 106–115

Table 2 ranging from 26.5, 21.4 and 16.1 nm by area, perimeter and dimensions
Amount and specific activity of proteins in each enzymes. respectively with a mean size of 21.3 nm. Geetha et al. [36] revealed
that zinc oxide nanoparticle synthesized using Euphorbia jatropa latex is
Enzyme Protein (μg) Activity (U) Specific activity (μg of
protein)−1 hexagonal in shape with average particles size of 100 nm. The size of
Pp-ZnO NPs in this study corroborates with findings of Divya et al. [37]
α-amylase 162.58 84.72 0.05 who reported that the size of ZnO NPs were between 30 and 56 nm. The
Cysteine protease 138.45 72.21 0.48
SAED pattern obtained in the present study revealed that the diffraction
α- glucosidase 124.65 70.30 0.26
β-glucosidase 128.44 68.28 0.38 rings of the synthesized ZnO exhibited Debye–Scherrer rings assigned to
Glutathione transferase 142.12 76.17 0.35 (100), (002), (101), (102), (110), (103) and (112) respectively. The
Lipase 118.65 64.70 0.36 particle size determined from TEM analysis is close to that of the XRD
Lactate dehydrogenase 92.58 46.34 0.25 analysis. The element composition of Pp-ZnO NPs was determined by
energy dispersive X-ray spectra (EDX). EDX spectrum showed that the
zinc in the composition of nanoparticle was 87.36%. This was in ac-
methods using different plant extracts is also possible. The green
cordance with the findings of Malaikozhundan et al. [8] who reported
synthesis of ZnO NPs is much safer and environment friendly compared
that the elemental composition of zinc in P. pinnata seed extract coated
to chemical synthesis because it does not lead to the formation of toxic
ZnO NPs was 84%. The magnitude of zeta potential (−30 mV to
byproducts. UV–vis spectroscopy is an analytical technique used to
+30 mV) gives an indication of the potential stability of the colloidal
determine the synthesis of ZnO NPs. In UV–vis spectroscopy, con-
system. In the present study, Pp-ZnO NPs are negatively charged and
ducting electrons start oscillating at a certain wavelength range due to
moderately stable with the zeta potential value of −12.45 mV. This was
surface plasmon resonance (SPR) effect. The UV–vis spectra of freshly
in agreement with findings of Selvarajan and Mohanasrinivasan [38]
prepared Pp-ZnO NPs exhibited maximum absorption peak (λmax) at
who reported that ZnO NPs are moderately stable with zeta value of
370 nm. The increase in the absorbance peak of Pp-ZnO NPs may be due
−15.3 mV.
to the functional molecules present in the leaf extract. This was in ac-
Application of nanomaterials can revolutionize agriculture by de-
cordance with the findings of Santhoshkumar et al. [32] who reported
veloping potential and effective methods for pest management [11]. It
that the zinc oxide nanoparticle synthesized using the leaf extract of
was reported that the pungam oil based gold nanoparticles (PO-AuNPs)
Passiflora caerulea showed strong absorbance spectra at 380 nm due to
significantly reduced the fecundity of Pericalia ricini [39]. P. pinnata
surface plasmon resonance. Similar observations have been docu-
seed methanolic extract reduced egg laying by Helicoverpa armigera
mented by Malaikozhundan et al. [8] who revealed that the zinc oxide
[40]. The oviposition deterrent activity of ethanolic extracts of P. pin-
nanoparticle synthesized using the seed extract of P. pinnata exhibited
nata against Aedes aegyptii and Culex quinquefasciatus has been reported
absorbance peak at 380 nm.
by Swathi et al. [41]. Similar to these reports, in the present study, egg
X-ray diffraction is an analytical technique used for the phase
laying by C. maculatus was significantly reduced after treatment with
identification of crystalline nanoparticle. In the present study, Pp-ZnO
Pp-ZnO NPs at 25 μg mL−1. A greater reduction in the fecundity po-
NPs showed various Bragg’s reflection peaks at 31.62°, 34.88°, 36.50°,
tential of C. maculatus was observed after treatment with Bt-ZnO NPs at
39.34°, 47.24°, 57.24°, 63.85°, 66.26° and 69.27° which corresponds to
25 μg/mL [16]. Neem oil was found to be the most effective in in-
the lattice planes at (100), (002), (101), (102), (110), (103), (200) and
creasing the developmental period of Lassioderma serricorne which was
(112) reflection lines of hexagonal ZnO NPs, respectively. The XRD
followed by karanj, lemongrass, mustard, citronella and groundnut oil
peaks obtained in the present study are consistent with the ZnO hex-
[42]. In the present study, the larval, pupal and total development
agonal phase (wurtzite structure) of standard JCPDS No.897102. The
period was significantly prolonged after treatment with Pp-ZnO NPs at
calculated size of the crystalline particle was found to be 19.4 nm. Our
25 μg mL−1. Treatment with PO-AuNPs significantly increased the
results corroborates with the observations of Malaikozhundan et al. [8]
larval period, pupal period and adult longevity of P. ricini [39]. Our
who reported that the average crystalline size of P. pinnata seed extract
results are further supported by the observations of Malaikozhundan
coated zinc oxide nanoparticles was 30.2 nm. Vijayakumar et al. [9]
et al. [16] who stated that the total development period (egg-larval-
demonstrated that the crystalline size of L. nobilis leaf extract coated
pupal period) of C. maculatus was significantly prolonged after treat-
zinc oxide nanoparticle was 24 nm.
ment with Bt-ZnO NPs.
FTIR spectroscopy was performed to identify the functional bio-
In recent times, nanoparticles have received much attention for
molecules responsible for capping and efficient stabilization of Pp-ZnO
controlling pests in agriculture [43,44,45]. Zinc nanoparticles have
NPs. The FTIR spectra of Pp-ZnO NPs exhibited peaks at 1754, 1655,
antimicrobial activity and can be used as fungicide [46,47]. The in-
1639, 1390, 1148 and 1086 cm−1. The intense broad band at 1754 and
secticidal activity of ZnO-TiO2-Ag nanoparticles on Western flower
1655 cm−1could be attributed to the stretching of polyphenols. The
thrips, Frankliniella occidentalis Pergande have been reported and most
band observed at 1639 and 1390 cm−1 could be due to the stretching of
mortality effect pertained to ZnO (28%), TiO2 (70%) and Ag (2%) [48].
aromatic amines. The band at 1148 cm−1 corresponds to CeC
In the present study, treatment with Pp-ZnO NPs at 25 μg mL−1 in-
stretching vibrations of alkanes. Band at 1086 cm−1 could be attributed
creased the mortality of C. maculatus to 100% with an estimated LC50 of
to CeN stretching vibrations of aliphatic amides. The FTIR spectra of P.
10.85 μg mL−1. These results are in agreement with the findings of
pinnata leaf extract showed intense broad band at 1735 cm−1 which
Malaikozhundan et al. [16] who revealed that the Bt-ZnO NPs produced
corresponds to hydroxyl groups. The band at 1640 and 1436 cm−1 was
100% mortality of C. maculatus with least LC50 value of 10.71 μg mL−1.
attributed to the CeOH group of alkanes. The band observed at 1250
The leaf oil of Cymbopogon schoenanthus, rich in piperitone, gave 90%
and 1132 cm−1 could be due to the vibrations of HeH and OH groups
mortality after 24 h at a concentration of 6.7 μL/L [49]. Plectranthus
respectively. The band at 1065 cm−1 could be due to the stretching of
grandifolius essential oil, predominantly (E)-myroxide, was shown to be
carboxyl groups. FTIR results obtained in the present study are con-
toxic to both adults and eggs of C. maculatus. Likewise, Cinnamomum
sistent with the observations of Vijayakumar et al. [33], Sangeetha
aromaticum bark oil, rich in cis-cinnamaldehyde, was insecticidal with
et al. [34] and Elumalai et al. [35] who reported that the stability of
an LC50 of 27.6 μg/cm2 after 24 h [50].
ZnO NPs is due to the capping agent of plant extract involved in na-
Amylases are glycosidases that catalyze the hydrolysis of α-D-1, 4-
noparticle synthesis.
glucosidic linkages of starch, glycogen and related α-D-1-4 glucan
The size, shape and morphology of nanoparticle were determined
consisting of two types of polymers, amylose and amylopectin.
through HR-TEM. In the present study, Pp-ZnO NPs were agglomerated
Amylases catalyze the hydrolysis of amylose to disaccharides and
and exhibited a hexagonal wurtzite structure with a particle size
monosaccharides (maltose and glucose). Several insects, especially

113
B. Malaikozhundan, J. Vinodhini
those that feed on starchy seeds during larval and/or adult stages, de-
pend on their α-amylases for survival. Research on starch digestion as a
target for control of starch-dependent insects has been stimulated in
recent years after results showing that α-amylase inhibitors from P.
vulgaris seeds are detrimental to the development of cowpea weevil, C.
maculatus and Azuki bean weevil, C. chinensis [51,52]. In the present
study, a significant reduction in the α-amylase activity of C. maculatus
was observed after treatment with Pp-ZnO NPs at 25 μg mL−1. α-amy-
lase activity of C. maculatus was significantly reduced after treatment
with Bt-ZnO NPs at 25 μg/mL [16]. Artemisia annua extract caused the
reduction of α-amylase activity in Pieris rapae L. [53].
In insects, the cysteine proteinases are utilized in the digestive
processes, but are found in several other tissues, indicating that they
may also play other roles [54,55]. In the present study, a significant
reduction in the cysteine protease activity of C. maculatus was observed
after treatment with Pp-ZnO NPs. Our results corroborates with the
findings by Malaikozhundan et al. [16] who demonstrated that the
cysteine protease activity of C. maculatus was dramatically decreased
after treatment with Bt-ZnO NPs at 25 μg/mL. Zibaee et al. [56] ob-
served that the biopesticide significantly decreased the activity of
proteases in H. cunea larvae.
Glucosidases catalyze the hydrolysis of 1, 4-linked α-D-glucose re-
sidues with release of α-D-glucose. The study of glucosidases in herbi-
vorous insects is important not only for understanding the biochemistry
of digestion but also for developing insect pest management strategies
[57]. In the present study, both α and β- glucosidase activity was sig-
nificantly reduced after treatment with Pp-ZnO NPs at 25 μg mL−1. This
was consistent with the reports of Malaikozhundan et al. [16] who
recorded a reduction in the α-glucosidase activity of C. maculatus after
treatment with Bt-ZnO NPs. Treatment of H. cunea larvae with sub le-
thal concentrations of different biopesticides exhibited a reduction in
the activity of α- and β-glucosidases [56]. Glutathione S-transferase
(GST) is an antioxidant enzyme that plays a major role in detoxification
[58]. Decreased GST activity of C. maculatus in the present study sug-
gests a poor defense mechanism in the detoxification of Pp-ZnO NPs.
Treatment with Bt-ZnO NPs decreased the activity of GST in C. macu-
latus [16]. The involvement of bruchid glutathione S-transferases in the
detoxification of insecticides and bioinsecticides has been reported by
Kolawole et al. [59]. Lipases (triacylglycerol acylhydrolase), catalyses
the hydrolysis of fatty acid ester bonds, and are widely distributed
among animals, plants and microorganisms [60]. Lipases play a major
role in storage and lipid mobilization. These enzymes are the basic
components in many of the physiological process like, reproduction,
growth, and defense against pathogens [29]. In the present study, a
significant reduction in the mid-gut lipase activity of C. maculatus was
observed after treatment with Pp-ZnO NPs. Treating the rice leaf folder,
Cnaphalocrocis medinali, with Bco (Bacillus thuringiensis Kurstaki), NSKE
(neem seed kernal), and VNLE (Vitex negundo leaf extract) (azadirachtin
and neem components) sharply decreased the activity level of lipase in
the mid-gut [61]. Zibaee et al. [56] found that B. thuringiensis and plant
extracts decreased the activity of lipase in H. cunea larvae. This study
concludes that Pp-ZnO NPs are highly effective against C. maculatus
than the leaf extract and uncoated ZnO NPs.
5. Conclusion
This study reports the green synthesis of P. pinnata leaf extract
coated zinc oxide nanoparticle (Pp-ZnO NPs) for the first time. The
green synthesized Pp-ZnO NPs were crystalline in nature with a mean
size of 21.3 nm. Pp-ZnO NPs showed significant effects on the fecundity,
hatchability, larval and pupal development period of C. maculatus. Use
of Pp-ZnO NPs delayed the total development period of C. maculatus. In
addition, Pp-ZnO NPs were highly effective in the control of C. macu-
latus with LC50 at 10.85 μg mL−1. Furthermore, 100% mortality of C.
maculatus was observed at 25 μg mL−1 of Pp-ZnO NPs. This study
concludes that Pp-ZnO NPs are highly effective against C. maculatus and
114
Materials Today Communications 14 (2018) 106–115
can be used as a promising control agent for the management of insect
pests of stored commodities in the future.
Conflict of interest
The authors declare no conflict of interest
Acknowledgements
Dr.B.Malaikozhundan gratefully acknowledges DST-SERB, New
Delhi for the support of a research grant under the Young Scientist
Scheme (YSS/2015/000645). Dr.B.Malaikozhundan thanks the
National Pulses Research Centre, Vamban, Pudukottai District, Tamil
Nadu, India for the supply of pulses.
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